The invention relates generally to the field of somatic cells and cell lines altered with respect to the expression of a gene detrimental to early development, and particularly relates to cells with an altered PrP gene.
Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not infect another (e.g., a mouse).
A major step in the study of prions and the diseases that they cause was the discovery and purification of a protein designated prion protein (xe2x80x9cPrPxe2x80x9d) [Bolton et al., Science 218:1309-11 (1982); Prusiner et al., Biochemistry 21:6942-50 (1982); McKinley et al., Cell 35:57-62 (1983)]. Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrPC is encoded by a single-copy host gene [Basler et al., Cell 46:417-28 (1986)] and is normally found at the outer surface of neurons. A leading hypothesis is that prion diseases result from conversion of PrPC into a modified form called PrPSc.
At present, it appears that the scrapie isoform of the prion protein (PrPSc) is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans. See Prusiner, S. B., xe2x80x9cMolecular biology of prion disease,xe2x80x9d Science 252:1515-1522 (1991). The most common prion diseases of animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith, J. and Wells, Microbiol. Immunol. 172:21-38 (1991)]. Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek, D. C., Science 197:943-960 (1977); Medori et al., N. Engl. J. Med. 326:444-449 (1992)]. The presentation of human prion diseases as sporadic, genetic and infectious illnesses initially posed a conundrum which has been explained by the cellular genetic origin of PrP.
Some cases of human prion disease have been transmitted to rodents but apparently with less regularity than transmission between animals of the same species [Gibbs, Jr. et al., Slow Transmissible Diseases of the Nervous System, Vol. 2, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 87-110 (1979); Tateishi et al., Prion Diseases of Humans and Animals, Prusiner et al., eds. (London: Ellis Horwood), pp. 129-134 (1992)]. The infrequent transmission of human prion disease to rodents has been cited as an example of the xe2x80x9cspecies barrierxe2x80x9d first described by Pattison in his studies of passaging the scrapie agent between sheep and rodents [Pattison, I. H., NINDB Monograph 2, D. C. Gajdusek, C. J. Gibbs Jr. and M. P. Alpers, eds. (Washington, D.C.: U.S. Government Printing), pp. 249-257 (1965)]. In those investigations, the initial passage of prions from one species to another was associated with a prolonged incubation time with only a few animals developing illness. Subsequent passage in the same species was characterized by all the animals becoming ill after greatly shortened incubation times.
The molecular basis for the species barrier between Syrian hamster (SHa) and mouse was shown to reside in the sequence of the PrP gene using transgenic (Tg) mice [Scott et al., Cell 59:847-857 (1989)]. SHaPrP differs from MoPrP at 16 positions out of 254 amino acid residues [Basler et al., Cell 46:417-428 (1986); Locht et al., Proc. Natl. Acad. Sci. USA 83:6372-6376 (1986)]. Tg(SHaPrP) mice expressing SHaPrP had abbreviated incubation times when inoculated with SHa prions. When similar studies were performed with mice expressing the human, or ovine PrP transgenes, the species barrier was not abrogated, i.e., the percentage of animals which became infected were unacceptably low and the incubation times were unacceptably long. Thus, it has not been possible, for example in the case of human prions, to use transgenic animals (such as mice containing a PrP gene of another species) to reliably test a sample to determine if that sample is infected with prions. Such a test was first disclosed in application Ser. No. 08/242,188 filed May 13, 1994 which is now U.S. Pat. No. 5,565,186 issued Oct. 15, 1996.
Most human CJD cases are sporadic, but about 10-15% are inherited as autosomal dominant disorders that are caused by mutations in the human PrP gene [Hsiao et al., Neurology 40:1820-1827 (1990); Goldfarb et al., Science 258:806-808 (1992); Kitamoto et al., Proc. R. Soc. Lond. 343:391-398 (1994)]. Iatrogenic CJD has been caused by human growth hormone derived from cadaveric pituitaries as well as dura mater grafts [Brown et al., Lancet 340:24-27 (1992)]. Despite numerous attempts to link CJD to an infectious source such as the consumption of scrapie infected sheep meat, none has been identified to date [Harries-Jones et al., J. Neurol. Neurosurg. Psychiatry 51:1113-1119 (1988)] except in cases of iatrogenically induced disease. On the other hand, kuru, which for many decades devastated the Fore and neighboring tribes of the New Guinea highlands, is believed to have been spread by infection during ritualistic cannibalism [Alpers, M. P., Slow Transmissible Diseases of the Nervous System, Vol. 1, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 66-90 (1979)].
More than 45 young adults previously treated with HGH derived from human pituitaries have developed CJD [Koch et al., N. Engl. J. Med. 313:731-733 (1985); Brown et al., Lancet 340:24-27 (1992); Fradkin et al., JAMA 265:880-884 (1991); Buchanan et al., Br. Med. J. 302:824-828 (1991)]. Fortunately, recombinant HGH is now used, although the seemingly remote possibility has been raised that increased expression of wt PrPC stimulated by high HGH might induce prion disease [Lasmezas et al., Biochem. Biophys. Res. Commun.196:1163-1169 (1993)]. That the HGH prepared from pituitaries was contaminated with prions is supported by the transmission of prion disease to a monkey 66 months after inoculation with a suspect lot of HGH [Gibbs, Jr. et al., N. Engl. J. Med. 328:358-359 (1993)]. The long incubation times associated with prion diseases will not reveal the full extent of iatrogenic CJD in thousands of people treated with HGH worldwide. Iatrogenic CJD also appears to have developed in four infertile women treated with contaminated human pituitary-derived gonadotrophin hormone [Healy et al., Br. J. Med. 307:517-518 (1993); Cochius et al., Aust. N. Z. J. Med. 20:592-593 (1990); Cochius et al., J. Neurol. Neurosurg. Psychiatry 55:1094-1095 (1992)] as well as at least 11 patients receiving dura mater grafts [Nisbet et al., J. Am. Med. Assoc. 261:1118 (1989); Thadani et al., J. Neurosurg. 69:766-769 (1988); Willison et al., J. Neurosurg. Psychiatric 54:940 (1991); Brown et al., Lancet 340:24-27 (1992)]. These cases of iatrogenic CJD underscore the need for screening pharmaceuticals that might possibly be contaminated with prions.
Two doctors in France were charged with involuntary manslaughter of a child who had been treated with growth hormones extracted from corpses. The child developed Creutzfeldt-Jakob Disease. (See New Scientist, Jul. 31, 1993, page 4). According to the Pasteur Institute, since 1989 there have been 24 reported cases of CJD in young people who were treated with human growth hormone between 1983 and mid-1985. Fifteen of these children have died. It now appears as though hundreds of children in France have been treated with growth hormone extracted from dead bodies at the risk of developing CJD (see New Scientist, Nov. 20, 1993, page 10.) In view of such, there clearly is a need for a convenient, cost-effective method for producing human products such as growth hormone that are free from any potentially contagious prion contamination.
The risk of transmitting prion-related disorders through therapeutic human products is a serious health concern. One method for preventing the transmission of prion related disorders is to produce recombinant human products in organisms such as Escherichia coli and Saccharomyces cerevisiae, since these organisms do not have an endogenous PrP gene and thus are not susceptible to PrPSc infection. While Escherichia coli and Saccharomyces cerevisiae production is ideal for the large scale synthesis of many human proteins, factors such as plasmid stability and insolubility of the desired protein product may limit the usefulness of these systems in some circumstances. In addition, certain recombinantly-produced proteins require post-translational modification to obtain the function of the endogenous protein, and thus may require synthesis in mammalian cells or even species-specific cell lines for proper functioning of the produced protein. For example, a recombinant human thryotropin (rhTSH) produced in Chinese Hamster Ovary cells is more highly sialylated than a nonrecombinant, cadaver-derived pituitary hTSH. The rhTSH also has a 2-fold lower metabolic clearance rate than pituitary TSH, resulting in a greater than 10-fold higher serum concentration of rhTSH compared to pituitary hTSH. (Thotakura et al., Endocrinology 128:341-348 (1991)) Since it is desirable to use therapeutic agents with the proper post-translational modifications, mammalian systems are preferable for the production of such proteins.
Moreover, other therapeutic agents, such as antibodies, are exclusively produced by mammalian cell systems. Classical cell fusion techniques allow efficient production of monoclonal antibodies by fusing the B cell producing the antibody with an immortalized mammalian cell line. The resulting cell line is called a hybridoma cell line. Applications of human antibodies produced by these hybridoma systems have promising potential in the area of cancer, immunodeficiencies, and other diseases involving an immune response. For instance, the apoptosis-inducing human monoclonal antibody SC-1 has been shown to cause a significant induction of apoptotic activity in eight patients with poorly differentiated stomach adenocarcinoma (Vollmers et al. Oncol Rep 5:549-552 (1998)). In another example, the antibody to HER2/neu has been shown to be a promising therapy for human breast cancer (Valero, (1998) Semin. Oncol. 5: 549-552). Monoclonal antibodies produced in murine hybridoma systems require an additional step of xe2x80x9chumanizingxe2x80x9d the antibodies to prevent the antibodies from being recognized as foreign epitopes (See e.g. Sato et al., (1994) Mol. Immunol. 31: 371-381). These systems are susceptible to prion infection, and antibodies produced in infected cells pose a risk of transmission to any individual receiving antibodies from the infected sources.
Since many therapeutics are produced in mammalian systems, there is a need for ensuring the safety of the products isolated from such systems. Given the potential for the transmission of disease when these therapeutics are extracted from tissue, there is a need for a method of producing therapeutics that are free from the risk of human disease-causing contaminants such as prions.
A method for producing mammalian therapeutics free from prion contamination, cells for use in such methods, and prion-free therapeutic formulations produced via the cells are disclosed. The invention comprises producing such therapeutics in somatic cells having a genome with an artificially altered PrP gene. The PrP gene in these cells may be ablated, or replaced by an exogenous inducible form of the PrP gene. Preferably, the cells of the invention are from mouse, rat, hamster, cow, sheep, horse, pig, dog, cat, chicken, more preferably from primates, and most preferably human. These cells may be derived from transgenic animals with an altered PrP gene, or the PrP may have been altered in the cell. Such cells are no longer susceptible to PrP infection (PrPSc), as infection requires an interaction between the infectious prion agent and the endogenous form of the protein (PrPC). Therapeutics produced by such a method include, but are not limited to, peptides, proteins, antibodies, antisense RNA molecules, ribozymes, viral vectors, and the like. Any of the therapeutics can be combined with a carrier to provide an appropriate pharmaceutical formulation which is prion free.
The invention features cells and a method of producing therapeutics using somatic mammalian cells in which the endogenous PrP gene has been disrupted and an exogenous PrP gene has been introduced into the genome. The endogenous PrP gene may be disrupted and the exogenous PrP later introduced into the cells, or the endogenous PrP gene may be disrupted by replacement of the endogenous PrP gene with the exogenous form of the PrP gene, e.g. by site-specific homologous recombination. In addition, the introduced PrP gene may or may not be integrated into the cell""s genome.
In one embodiment, the invention features the production of therapeutics in host cells expressing exogenous PrP sequences from a species genetically diverse from the host cells. These cells express PrPC, but are protected from prion infection from the host cell species-specific PrPSc.
In another embodiment, the invention features the production of therapeutics in host cells expressing exogenous PrP sequences of the same species as the host cell, with the endogenous form of PrP ablated.
Another aspect of the invention is a method of producing antibodies in a hybridoma cell with a disrupted endogenous PrP gene. The disruption of the PrP gene may follow the fusion of the antibody-producing B-cell with the immortalized cell line, and may occur either prior to the establishment of the hybridoma as a cell line or following establishment. Alternatively, the prion-free hybridoma cell line may be produced by transfecting B cells from animals with a disrupted PrP gene, thereby ablating the endogenous PrP gene of the B cell. These transfected B cells can be fused with an immortalized cell line which also has an ablated PrP gene, resulting in a hybridoma with no endogenous PrP expression that is resistant to prion infection.
Another aspect of the invention is the adaption of monoclonal antibodies for use of therapeutics by alteration and subsequent production in mammalian cells which cells have a disrupted endogenous PrP. Again, the endogenous PrP gene may be ablated or replaced with an inducible form of the PrP gene. Preferably, the therapeutic produced is a human antibody, and the antibody is xe2x80x9chumanizedxe2x80x9d in a PrP knock-out primate cell line.
An object of the invention is to provide a method for producing biological products that are free from the risk of prion infection, and thus will not transmit prion-related disorders to subjects receiving such products.
Another object of the invention is to provide a method for ensuring both bioactivity and safety of mammalian therapeutics.
A feature of the invention is that the cells used in the invention are not susceptible to prion infection.
An advantage of the invention is that the method ensures that biologic products created by this method are prion free.
An object is to provide a range of therapeutics and formulations thereof which are prion-free.
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.
Before the present cell lines, methods and prion-free products are described, it is to be understood that this invention is not limited to particular cell lines, methods, or products described and may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms xe2x80x9caxe2x80x9d, xe2x80x9can,xe2x80x9d and xe2x80x9cthexe2x80x9d include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to xe2x80x9ca constructxe2x80x9d includes a plurality of such constructs and reference to xe2x80x9ca mammalian cellxe2x80x9d includes reference to one or more mammalian cells, cell lines and equivalents thereof known to those skilled in the art, and so forth.
All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the cell lines, constructs, and methodologies that are described in the publications which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention. Further, the publication dates provided may be different from the actual publication date which may require independent verification.
The term xe2x80x9cisolatedxe2x80x9d shall mean separated away from its natural environment. An isolated protein is not necessarily separated away from all materials it is normally present with and may remain glycosylated.
The terms xe2x80x9ctherapeuticxe2x80x9d or xe2x80x9ctherapeutic agentxe2x80x9d as used herein generally mean any chemical or biological molecule used to obtain a desired pharmacologic, biologic, physiologic and/or psychologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. Therapeutics as used herein covers any compound used in the treatment of a disease in a mammal, particularly a human, and includes compositions for:
(a) preventing a disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it;
(b) inhibiting a disease symptom, i.e., arresting its development; or
(c) relieving a disease symptom, i.e., causing regression of the disease.
The term xe2x80x9ctreatmentxe2x80x9d is used herein to mean administering a xe2x80x9ctherapeuticxe2x80x9d to obtain all or any of the desired results of a xe2x80x9ctherapeutic.xe2x80x9d
A xe2x80x9cknock-outxe2x80x9d or xe2x80x9cablationxe2x80x9d of a gene, which terms are used interchangeably herein, means an alteration in the sequence of the gene or sequence associated with the gene that results in a decrease of function of the target gene, preferably such that target gene expression is undetectable or insignificant. An ablation of an endogenous PrP gene means that the function of any endogenous PrP gene has been substantially decreased so that expression is not detectable or only present at insignificant levels. xe2x80x9cKnock-outxe2x80x9d transgenics can be transgenic animals having a heterozygous knock-out of the PrP gene (PrP+/0) or a homozygous knock-out of the PrP gene (PrP0/0). xe2x80x9cKnock-outsxe2x80x9d also include conditional knock-outs, where alteration of the target gene can occur upon, for example, exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g., Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally.
The term xe2x80x9cPrnp-0/0xe2x80x9d or xe2x80x9cPrnp-Ablxe2x80x9d refers to a transgenic animal which has its PrP gene ablated with the xe2x80x9c0/0xe2x80x9d indicating that both alleles are ablated whereas 0/+indicates only one is ablated. Specifically, the animal being referred to is generally a transgenic mouse which has its PrP gene ablated i.e., a PrP knock-out mouse. In that the PrP gene is disrupted no mouse PrP protein is expressed.
The term xe2x80x9cprionxe2x80x9d shall mean an infectious particle known to cause diseases (spongiform encephalopathies) in humans and animals. The term xe2x80x9cprionxe2x80x9d is a contraction of the words xe2x80x9cproteinxe2x80x9d and xe2x80x9cinfectionxe2x80x9d and the particles are comprised largely if not exclusively of PrPSc molecules encoded by a PrP gene which expresses PrPC which changes conformation to become PrPSc. Prions are distinct from bacteria, viruses and viroids. Known prions include those which infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of sheep and goats as well as bovine spongiform encephalopathies (BSE) or mad cow disease and feline spongiform encephalopathies of cats. Four prion diseases known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI). As used herein prion includes all forms of prions causing all or any of these diseases or others in any animals used xe2x80x94and in particular in humans and in domesticated farm animals. Prions include all infectious variants of the PrPSc protein.
The terms xe2x80x9cPrP genexe2x80x9d and xe2x80x9cprion protein genexe2x80x9d are used interchangeably herein to describe genetic material which expresses PrP proteins. The term xe2x80x9cPrP genexe2x80x9d refers generally to any gene of any species which encodes any form of a PrP amino acid sequence including any prion protein. Some commonly known PrP sequences are described in Gabriel et al., Proc. Natl. Acad. Sci. USA 89:9097-9101 (1992) which is incorporated herein by reference to disclose and describe such sequences.
The terms xe2x80x9cstandardized prion preparation,xe2x80x9d xe2x80x9cprion preparation,xe2x80x9d xe2x80x9cpreparationxe2x80x9d and the like are used interchangeably herein to describe composition containing prions which composition is obtained from brain tissue of mammals which contain substantially the same genetic material as relates to PrP proteins, e.g., brain tissue from a set of mammals which exhibit signs of prion disease which mammals may comprise any of (1) a PrP chimeric transgene; (2) an ablated endogenous PrP gene; (3) a high copy number of PrP genes from a genetically diverse species; or (4) hybrids with an ablated endogenous PrP gene and a PrP gene from a genetically diverse species. The mammals from which standardized prion preparations are obtained exhibit clinical signs of CNS dysfunction as a result of inoculation with prions and/or due to their genetically modified make up, e.g., high copy number of PrP genes.
The terms xe2x80x9cablated PrP protein gene,xe2x80x9d xe2x80x9cdisrupted PrP gene,xe2x80x9d xe2x80x9cablated PrP gene,xe2x80x9d xe2x80x9cPrP%xe2x80x9d and the like are used interchangeably herein to mean an endogenous PrP gene which has been altered (e.g., add and/or remove nucleotides) in a manner so as to render the gene inoperative. Examples of nonfunctional PrP genes and methods of making such are disclosed in Bxc3xceler, H., et al. xe2x80x9cNormal development of mice lacking the neuronal cell-surface PrP protein,xe2x80x9d Nature 356:577-582 (1992) which is incorporated herein by reference. Both alleles of the genes are disrupted.
The terms xe2x80x9cresistant to infectionxe2x80x9d, xe2x80x9cresistant to infection with prionsxe2x80x9d and the like mean the cells include an altered PrP gene which renders the cells resistant to prion disease when inoculated with an amount and type of prion which would be expected to cause prion disease should the exposed cells or a product of the exposed cells be introduced in an animal of the same species.
The term xe2x80x9cprion-freexe2x80x9d means the composition contains an insufficient amount of prions (PrPSc) to cause an infection and preferably contains no detectable amount of prions using current detection technology and most preferably contains no prion at all.
The terms xe2x80x9csusceptible to infectionxe2x80x9d and xe2x80x9csusceptible to infection by prionsxe2x80x9d and the like are used interchangeably herein to describe cells which can be infected by prions, such infected cells being able to cause a subject animal to develop a disease if inoculated with these infected cells or products produced in such cells.
The term xe2x80x9cincubation timexe2x80x9d shall mean the time from inoculation of an animal with a prion until the time when the animal first develops detectable symptoms of disease resulting from the infection. A reduced incubation time is six months or less, preferably about 75 daysxc2x125 days or less, more preferably about 30 daysxc2x110 days or less.
The terms xe2x80x9cgenetically diverse animalxe2x80x9d and xe2x80x9cgenetically diverse mammalxe2x80x9d are used to describe an animal which includes a native PrP codon sequence which differs from the genetically diverse test animal by 17 or more codons, preferably 20 or more codons, and most preferably 28-40 codons from the host cell. Thus, a mouse PrP gene is genetically diverse with respect to the PrP gene of a human, cow or sheep, but is not genetically diverse with respect to the PrP gene of a hamster.
The term xe2x80x9cantibodyxe2x80x9d stands for an immunoglobulin protein which is capable of binding an antigen. Antibody as used herein is meant to include the entire antibody as well as any antibody fragments (e.g. F(abxe2x80x2, Fab, Fv) capable of binding the epitope, antigen or antigenic fragment of interest. Preferred antibodies for assays of the invention are immunoreactive or immunospecific for and therefore specifically and selectively bind to a PrP protein. Antibodies which are immunoreactive and immunospecific for both native PrPC and treated PrPSc but not native PrPSc are preferred. Antibodies for PrP are preferably immunospecificxe2x80x94e.g., not substantially cross-reactive with related materials. The term xe2x80x9cantibodyxe2x80x9d encompasses all types of antibodies, e.g. polyclonal, monoclonal, and those produced by the phage display methodology. Particularly preferred antibodies of the invention are antibodies which have a relatively high degree of affinity for the target antigen.
xe2x80x9cPurified antibodyxe2x80x9d refers to that which is sufficiently free of other proteins, carbohydrates, and lipids with which it is naturally associated. Such an antibody xe2x80x9cpreferentially bindsxe2x80x9d to a treated or denatured PrPSc protein (or an antigenic fragment thereof), and does not substantially recognize or bind to other antigenetically unrelated molecules. A purified antibody of the invention is preferably immunoreactive with and immunospecific for a specific species.
xe2x80x9cAntigenic fragmentxe2x80x9d of a protein (e.g., HER2/neu) means a portion of such a protein which is capable of binding an antibody.
By xe2x80x9cbinds specificallyxe2x80x9d is meant high avidity and/or high affinity binding of an antibody to a specific polypeptide e.g., epitope of a protein such as a HER2/neu protein. Antibody binding to the epitope on this specific polypeptide is preferably stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest e.g., binds more strongly to epitope fragments of a protein such as HER2/neu so that by adjusting binding conditions the antibody binds almost exclusively to an epitope site or fragments of a desired protein such as an epitope fragment exposed by treatment of HER2/neu and not exposed on related receptors of the same subfamily.
Preferably, the product of the PrP gene in cells used in the method of the invention is undetectable, insignificant, and most preferably non-existent. A knock-out of an endogenous PrP gene means that the function of the PrP protein has been substantially decreased so that PrP protein expression is not detectable or only present at insignificant levels. This may be achieved by a variety of mechanisms, including introduction of a disruption of the coding sequence, e.g. insertion of one or more stop codons, insertion of a DNA fragment, deletion of coding sequence, substitution of stop codons for coding sequence, etc. In some cases the exogenous transgene sequences are ultimately deleted from the genome, leaving a net change to the native sequence. Different approaches may be used to achieve the xe2x80x9cknock-out.xe2x80x9d See U.S. Pat. Nos. 5,464,764, 5,627,059 and related patents and publications to Capecchi et al. A chromosomal deletion of all or part of the native gene may be induced, including deletions of the non-coding regions, particularly the promoter region, 3xe2x80x2 regulatory sequences, enhancers, or deletions of gene that activate expression of PrP genes. A functional knock-out may also be achieved by the introduction of an anti-sense construct that blocks expression of the native genes (for example, see Li and Cohen (1996) Cell 85:319-329). xe2x80x9cKnock-outsxe2x80x9d also include conditional knock-outs, for example where alteration of the target gene occurs upon exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g. Cre in the Cre-lox system), or other methods for directing the target gene alteration.
In general, site-specific recombination-facilitating sequences useful in the present invention encompass any nucleotide sequence that facilitates site-specific recombination by interaction of a specific enzyme with two such site-specific recombination-facilitating sequences. Exemplary site-specific recombination facilitating sequences include, but are not necessarily limited to: PNS vectors as described in U.S. Pat. No. 5,627,059; lox sequences (recombination mediated by Cre enzyme); frt sequences (Golic et al. (1989) Cell 59:499-509 and O""Gorman et al. (1991) Science 251:1351-5); recombination mediated by the FLP recombinase; the recognition sequences for the pSR1 recombinase of Zygosaccharomyces rouxii (Matsuzaki et al. (1990) J. Bacteriol. 172:610-8); and the like. Each of these can be used to alter endogenous PrP expression by disrupting the endogenous gene, i.e. creating a PrP knock-out, and/or by replacing the endogenous gene with an inducible form of PrP, i.e. creating a conditional PrP knock-out.
The exogenous introduced PrP gene may be a mammalian PrP gene which is operably linked to an inducible promoter. By xe2x80x9coperably linkedxe2x80x9d is meant that a DNA sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules, e.g. transcriptional activator proteins, are bound to the regulatory sequence(s). Such an inducible PrP gene functions as a conditional knock-out, since induction of PrP can be reversibly controlled.
Specific constructs of interest include, but are not limited to, anti-sense PrP, which will block native PrP expression, expression of dominant negative PrP mutations, and over-expression of a PrP gene. A detectable marker, such as lac Z may be introduced into the locus, where upregulation of expression will result in an easily detected change in phenotype. Constructs utilizing the PrP promoter region, in combination with a reporter gene or with the coding region, are also of interest.
DNA constructs for homologous recombination will comprise at least a portion of the PrP gene with the desired genetic modification, and will include regions of homology to the target locus. DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection are included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al. (1990) Methods in Enzymology 185:527-537.
The term xe2x80x9cPrPxe2x80x9d is used generically to designate PrP genes, e.g. homologs from rat, human, mouse, guinea pig, etc., and their alternate forms. Used generically, this term encompasses different isoforms, polymorphisms, variant sequences, and mutated forms of PrP as well. The term is also intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5xe2x80x2 and 3xe2x80x2 non-coding nucleotide sequences involved in the regulation of expression, up to about 1 kb beyond the coding region, but possibly further in either direction. The DNA sequences encoding PrP may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host. The amino acid sequences and DNA sequences for a number of animals are known, see U.S. Pat. Nos. 5,565,186; 5,763,740; 5,789,655; 5,792,901 and publications cited in these patents for sequences, isoforms, polymorphisms, variants and mutations.
A genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the sequences listed here and in the cited patents and publications, including all of the introns that are normally present in a native chromosome. It may further include the 3xe2x80x2 and 5xe2x80x2 untranslated regions found in the mature mRNA. It may farther include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5xe2x80x2 or 3xe2x80x2 end of the transcribed region. The genomic DNA may be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence.
The sequence of this 5xe2x80x2 region, and further 5xe2x80x2 upstream sequences and 3xe2x80x2 downstream sequences, may be utilized for promoter elements, including enhancer binding sites, that provide for expression in tissues where PrP is expressed. The tissue specific expression is useful for determining the pattern of expression, and for providing promoters that mimic the native pattern of expression. Naturally occurring polymorphisms in the promoter region are useful for determining natural variations in expression, particularly those that may be associated with disease. Alternatively, mutations may be introduced into the promoter region to determine the effect of altering expression in experimentally defined systems. Methods for the identification of specific DNA motifs involved in the binding of transcriptional factors are known in the art, e.g. sequence similarity to known binding motifs, gel retardation studies, etc. For examples, see Blackwell et al. (1995) Mol Med 1:194-205; Mortlock et al. (1996) Genome Res. 6:327-33; and Joulin and Richard-Foy (1995) Eur J Biochem 232:620-626.
The regulatory sequences may be used to identify cis acting sequences required for transcriptional or translational regulation of PrP expression, especially in different tissues or stages of development, and to identify cis acting sequences and trans acting factors that regulate or mediate expression. Such transcription or translational control regions may be operably linked to a PrP gene in order to promote or prevent expression of wild type or altered PrP or other proteins of interest in cultured cells.
The nucleic acid compositions used in the subject invention may encode all or a part of the PrP polypeptides as appropriate. Fragments may be obtained of the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. For the most part, DNA fragments will be of at least 15 nt, usually at least 18 nt, more usually at least about 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening, etc. Larger DNA fragments, i.e. greater than 100 nt are useful for production of the encoded polypeptide. For use in amplification reactions, such as PCR, a pair of primers will be used.
Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for scanning mutations may be found in Gustin et al., 1993 Biotechniques 14:22; Barany, 1985 Gene 37:111-23; Colicelli et al., 1985 Mol Gen Genet 199:537-9; and Prentki et al., 1984 Gene 29:303-13. Methods for site specific mutagenesis can be found in Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, CSH Press, pp. 15.3-15.108; Weiner et al., 1993 Gene 126:35-41; Sayers et al., 1992 Biotechniques 13:592-6; Jones and Winistorfer, 1992 Biotechniques 12:528-30; Barton et al., 1990 Nucleic Acids Res 18:7349-55; Marotti and Tomich, 1989 Gene Anal Tech 6:67-70; and Zhu 1989 Anal Biochem 177:120-4. For example, a chicken, bovine, sheep, rat and mouse PrP gene are disclosed and published within Gabriel et al., Proc. Natl. Acad. Sci. USA 89:9097-9101 (1992). The sequence for the Syrian hamster is published in Basler et al., Cell 46:417-428 (1986). The PrP gene of sheep is published by Goldmann et al., Proc. Natl. Acad. Sci. USA 87:2476-2480 (1990). The PrP gene sequence for bovine is published in Goldmann et al., J. Gen. Virol. 72:201-204 (1991). The sequence for chicken PrP gene is published in Harris et al., Proc. Natl. Acad. Sci. USA 88:7664-7668 (1991). The PrP gene sequence for mink is published in Kretzschmar et al., J. Gen. Virol. 73:2757-2761 (1992). The human PrP gene sequence is published in Kretzschmar et al., DNA 5:315-324 (1986). The PrP gene sequence for mouse is published in Locht et al., Proc. Natl. Acad. Sci. USA 83:6372-6376 (1986). The PrP gene sequence for sheep is published in Westaway et al., Genes Dev. 8:959-969 (1994). These publications are all incorporated herein by reference to disclose and describe the PrP gene and PrP amino acid sequences.
The tetracycline (tet)-regulated trans-activation systems for inducible gene expression allows temporal and quantitative control of exogenous genes in mammalian cells, transgenic mice and plants. For review, see Shockett, PNAS 43:5173-5176 (1996), which is incorporated by reference herein. The pioneering tet-regulated gene expression system involved a constitutive expression of the tet transactivator protein (tTA) with the cytomegalovirus (CMV) immediate early (IE) promoter/enhancer. tTA is a fusion protein composed of the tet repressor of Escheria coli and the transcriptional activation domain of the VP16 protein of herpes simplex virus. In the absence of tetracycline, the tet repressor portion of tTA mediates high affinity binding to sequences from the tet resistance operator of Tn10 (tetO). In the presence of tetracycline, a conformational change in the tet repressor prevents tTA from binding to its operator.
A modified system has also been developed using a reverse transactivator (rtTA) that binds tetO efficiently only in the presence of the tet derivatives doxycycline or anhydrotetracycline. It is hypothesized that this system is especially useful in situations where cells or individuals were to be kept in the repressed state for long periods of time and where long term exposure to tet or one of its derivatives was undesirable, or in situations requiring rapid induction.
Although the preferred embodiment of the present invention feature a tetracycline-inducible system driven by the CMV promoter, other methods of delivery of the tet-regulated genes and other resistant factors may be used. For example, viral vectors driven by either the SV40 promoter, by glial-cell specific promoters, or by the autonomous parvovirus LuIII may be used to express tTA. These and other similar systems may be used in the present invention without departing from the spirit of the disclosure, as will be obvious to those skilled in the art. For example, systems such as ecdysome inducible systems can be used instead of the tetracycline inducible system.
Antibodies are prepared in accordance with conventional methods, where the expressed polypeptide or protein is used as an immunogen, by itself or conjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the like. Various adjuvants may be employed, with a series of injections, as appropriate. For monoclonal antibodies, after one or more booster injections, the spleen is isolated, the lymphocytes immortalized by cell fusion, and then screened for high affinity antibody binding. The immortalized cells, i.e. hybridomas, producing the desired antibodies may then be expanded. For further description, see Monoclonal Antibodies: A Laboratory Manual, Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1988.
Once a suitable monoclonal antibody has been identified as a potential therapeutic agent, the antibody must be adapted for use in the subject organism. Altering the antibody to conform to the immune system of the subject species serves two basic functions: it decreases the chance of a significant adverse reaction of the host immune response to the therapeutic antibody, and it increases the therapeutic activity of the antibody since the activity is less likely to be neutralized by the host immune system. For example, antibodies used as human therapeutics are routinely xe2x80x9chumanizedxe2x80x9d before used to treat human ailments.
There are many approaches to humanizing monoclonal antibodies, each of which designs and constructs a reshaped human antibody which mimics the mouse antibody. For instance, one approach bases the design of the human antibody on the most homologous consensus sequence. Another approach bases the design on the most homologous human antibody. Both of these approaches utilize primate cell cultures, and therefore bear the risk of contamination with PrPSc and potential infection of people treated with such therapeutics. Elimination of the endogenous PrP gene in the cell lines used to humanize these antibodies can prevent this occurrence.